1. Field of the Invention
The present invention relates to a method of maintaining or improving the activity of industrially useful nitrile hydratase. The activity referred to herein is a nitrile hydrating activity determined by a method described in the Examples.
2. Description of the Related Art
The nitrile hydrating activity of nitrile hydratase is unstable and easily reduced with time. For preventing such reduction in the nitrile hydrating activity of nitrile hydratase with time to maintain the activity, a method which comprises adding at least one compound selected from a nitrile, an amide, an organic acid and a salt thereof as a stabilizer to a suspension or solution of a nitrile hydratase-containing microorganism or a treated material of the microorganism is disclosed (see, for example, JP-B 5-43351 and JP-B 4-48435).
A storage method wherein a microorganism or a treated microbial material is in a state suspended in an aqueous medium and the aqueous medium is neutral or weakly basic and having dissolved therein an inorganic salt at a concentration of 100 mM to its saturated concentration is also disclosed (see, for example, JP-A 8-112089). However, both the methods are effective only in maintaining a nitrile hydratase activity, and an effect of further increasing the nitrile hydratase activity is not reported therein.
As a method of increasing a nitrile hydrating activity by producing a nitrile hydratase-containing microorganism through culture and then subjecting the resulting microorganism or a treated material of the microorganism to some treatment, a method which comprises irradiating the resulting Gram-positive microorganism having a nitrile hydratase activity with light is disclosed (see, for example, JP-B2-35). However, this method is effective only in increasing the nitrile hydratase activity of the microorganism obtained through culture, and does not improve the nitrile hydratase activity of a microorganism whose activity was reduced. In addition, this method is irrelevant to maintenance of nitrile hydratase activity.
It is also reported that oxygen is necessary for culturing a nitrile hydratase-containing microorganism, and the concentration of dissolved oxygen during culture is maintained in the range of from 1 ppm to its saturated concentration, whereby the growth of the microorganism can be improved, and simultaneously the microorganism expressing a nitrile hydratase activity at high levels can be obtained (see, for example, JP-A 2002-17339). However, it is merely reported in this method that during culture, that is, under conditions where the nitrile hydratase-containing microorganism grows, there is the optimum concentration of dissolved oxygen in a culture solution in order to express a nitrile hydratase activity, and there is no description therein of maintenance and improvement of the nitrile hydratase activity under conditions where the nitrile hydratase-containing microorganism does not grow, that is, after the conclusion of culture.
The detailed mechanism of expression and reduction of nitrile hydratase activity is not completely elucidated, but it is described that for example, the reduction of the nitrile hydrating activity of iron-type nitrile hydratase derived from Rhodococcus sp. N-771 is attributable to oxidation, with oxygen in the air, of cysteinesulfenic acid coordinated with non-heme iron (III) as central metal into cysteinesulfinic acid (see, for example, M. Okada, M. Tsujimura and I. Endo: RIKEN Review, No. 41, p. 58-60 (2001)).
It is also described that for example, the activity of iron-type nitrile hydratase derived from Rhodococcus sp. N-771 is lost when trivalent iron as central metal is reduced to divalent iron by 2-cyano-2-propylhydroxy peroxide while cysteinesulfenic acid is oxidized into cysteinesulfinic acid (see, for example, M. Okada, M. Tsujimura and I. Endo: New Development of Reaction in Organic Chemistry (in Japanese), No. 3, p. 17-20 (2001).
It is also described that the structure of nitrile hydratase with cobalt as central metal, derived from Rhodococcus rhodochrous J1, is not determined, but is estimated to be similar to that of iron nitrile hydratase (see, for example, P. K. Mascharak, Coordination Chemistry Reviews, No. 225, p. 201-214 (2002)).